Humanized anti-Tn-MUC1 antibodies and their conjugates

Abstract

Humanized anti-Tn-MUC1 antibodies and conjugates thereof. Conjugates comprising pyrrolobenzodiazepines (PBDs) having a labile protecting group in the form of a linker to the antibody are described.

The invention claimed is: 
     
       1. An isolated humanized antibody that binds to Tn-MUC1, wherein the isolated humanized antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, a light chain variable region having the amino acid sequence of SEQ ID NO: 31, 32, 33, or 34, and optionally comprises a constant region derived from one or more human antibodies. 
     
     
       2. The isolated humanized antibody according to  claim 1 , wherein the isolated humanized antibody comprises:
 (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (ii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (iii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (iv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (v) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (vi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (vii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (viii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (ix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (x) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 31; 
 (xxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xl) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xli) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xlii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xliii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xliv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xlv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xlvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xlvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xlviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (xlix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (l) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (li) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (lii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (liii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (liv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (lv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (lvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (lvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (lviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; 
 (lix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 33; 
 (lxxxviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (lxxxix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xc) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xci) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xcii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 5 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 6 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xciv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xcv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xcvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xcvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 10 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xcviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (xcix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (ci) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (ciii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (civ) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cvii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cviii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 22 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cx) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cxi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cxii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cxiii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cxiv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; 
 (cxv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 28 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34; or 
 (cxvi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 34. 
 
     
     
       3. The humanized antibody according to  claim 1 , wherein said antibody binds Tn-MUC1 with an affinity (Kd) of at least 10 −6  M. 
     
     
       4. The humanized antibody according to  claim 3 , wherein said antibody binds Tn-MUC1 with an affinity (Kd) of at least 10 −9  M. 
     
     
       5. The humanized antibody according to  claim 1 , wherein said antibody competitively inhibits the binding to Tn-MUC1 of an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30. 
     
     
       6. The humanized antibody according to  claim 1 , wherein said antibody competitively inhibits the binding to Tn-MUC1 of the mouse 5E5 antibody. 
     
     
       7. The humanized antibody according to  claim 1 , wherein said antibody or antibody fragment substantially neutralizes at least one activity of at least one Tn-MUC1. 
     
     
       8. The humanized antibody according to  claim 1 , wherein said antibody or antibody fragment expresses at a level of at least 10 micrograms/ml in a static HEK293T transient expression system. 
     
     
       9. The humanized antibody according to  claim 1 , wherein said antibody or antibody fragment expresses at a level of at least 20 micrograms/ml in a shaken HEK293T transient expression system. 
     
     
       10. The humanized antibody according to  claim 1 , wherein said antibody has a melting temperature (T m ) of at least 62° C. 
     
     
       11. The humanized antibody according to  claim 1 , wherein said antibody or antibody fragment has a constant region of either isotype IgG1, IgG2, IgG3 or IgG4, or a mutated IgG constant region, and optionally a light chain constant region of isotype kappa or lambda. 
     
     
       12. The humanized antibody according to  claim 1 , wherein the humanized antibody fragment is a scFv, Fab or F(ab′) 2 . 
     
     
       13. A polynucleotide encoding a humanized antibody according to  claim 1 . 
     
     
       14. A conjugate comprising the humanized antibody according to  claim 1  coupled to a functional moiety. 
     
     
       15. The conjugate according to  claim 14 , wherein the functional moiety is selected from a drug, a reporter, a toxin, an organic moiety, and a binding member. 
     
     
       16. The conjugate according to  claim 15  wherein the reporter is a fluorescent compound, a radionuclide, or an enzyme. 
     
     
       17. The conjugate according to  claim 15  wherein the binding member is an antibody or antibody fragment. 
     
     
       18. The conjugate according to  claim 14 , wherein the humanized antibody is covalently bonded to the functional moiety. 
     
     
       19. A pharmaceutical composition comprising the humanized antibody according to  claim 1 , and a pharmaceutically acceptable diluent, carrier or excipient. 
     
     
       20. The pharmaceutical composition of  claim 19  further comprising a therapeutically effective amount of a chemotherapeutic agent. 
     
     
       21. A method of treating cancer comprising administering to a patient the pharmaceutical composition according to  claim 19 . 
     
     
       22. The method of  claim 21  wherein the patient is administered a chemotherapeutic agent, in combination with the humanized antibody. 
     
     
       23. A method of treating a proliferative disease in a subject, wherein the proliferative disease is associated with raised levels of Tn-MUC1, the method comprising administering to the subject the conjugate according to  claim 14 .